Translation of the RNA-encoded genetic message into the polypeptide chain of a protein links genotype to phenotype. It is carried out by the ribosome, an ancient ribonucleoprotein particle whose structural core and fundamental mechanism of action are conserved among all forms of life (C. R. Woese, et al. Microbiol. Rev. 47, 621 (1983); W. E. Hill, et al. Eds., The Ribosome. Structure, Function and Evolution (American Society for Microbiology, Washington D.C., (1990)). The smallest and best-studied examples are bacterial ribosomes, which have a molecular size of ˜2.5 MD and are made up of a small (30S) and a large (50S) subunit. The 30S subunit is composed of 16S rRNA (˜1500 nucleotides (nt)) and about 20 different proteins, whereas the large subunit contains 23S rRNA (˜2900 nt), 5S rRNA (120 nt), and more than 30 different proteins. This degree of structural complexity is in keeping with that of its biological role.
The substrate of the ribosome is tRNA, which is commonly considered to bind to the ribosome at three different sites: A, P, and E (aminoacyl, peptidyl, and exit, respectively) (Watson 1964; Rheinberger et al. 1981). Each tRNA binding site is partitioned between the two ribosomal subunits, resulting in as many as six different sites of interaction between tRNA and the ribosome. The anticodon ends of the tRNAs bind to the 30S subunit, which also carries messenger RNA (mRNA); the 3′-acceptor, or CCA ends of the tRNAs interact with the 50S subunit, which contains the catalytic site for peptide bond formation, peptidyl transferase (Monro 1967). Thus, the tRNAs span the interface between the 30S and 50S subunits.
The translational elongation cycle depends on three fundamental processes: (i) aminoacyl-tRNA selection, (ii) peptide bond formation, and (iii) translocation of tRNAs from one site to the next within the ribosome. Although in vivo, the steps of tRNA selection and translocation involve the elongation factors EF-Tu and EF-G, respectively, in guanosine triphosphate (GTP)-dependent reactions, both steps can be carried out by the ribosome in a factor-independent manner, under appropriate ionic conditions in vitro (Pestka 1969; Gavrilova et al. 1972). Thus, all three of the fundamental steps of the translation elongation cycle must be based on the properties of the ribosome itself, and most likely on its RNA components (Green et al. 1997). The molecular mechanisms by which the ribosome accomplishes these functional processes remain largely mysterious, as does its molecular structure. While knowledge of ribosome structure may not provide immediate explanations for the complexities of translation, it is clear that deeper mechanistic insights will depend on it.
Structures of ribosomal proteins and rRNA fragments, determined by x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, have provided atomic-resolution detail of individual components of the ribosome (Ramakrishnan et al. 1998; Moore et al. 1998; Nikonov et al. 1988; Szewcazk et al. 1995; Dallas et al. 1997; Correll et al. 1997). In recent years, great progress has been made in determining the structures of complete ribosomes, ribosomal subunits, and functional complexes of the ribosome by cryoelectron microscopy (Frank et al. 1995a; Stark et al. 1997a; reviewed in Agrawal et al. 1999a). Two major advances toward x-ray crystallography of the ribosome were the crystallization of 50S subunits (Yonath et al. 1980; von Bohlen et al. 1991) and the recent determination of their crystal structure at 9 Å resolution (Ban et al. 1998). Even more recently, two papers describing the structures of the T. thermophilus 30S ribosomal subunit at 5.5 Å resolution (Clemons et al. 1999) and the Haloarcula marismortui 50S ribosomal subunit at 5 Å resolution (Ban et al. 1999) were published. Although many of the details of the rRNA and ribosomal protein components are more clearly resolved in the subunit structures, some features seen in the 70S ribosome structure, such as protein L1 in the 50S subunit and part of the head of the 30S subunit, appear to be absent in the subunit maps, possibly because of local disorder that is not present in the 70S ribosome crystals. A 2.4 Angstrom structure of the 50S ribosomal subunit from Haloarcula marismortui was recently reported (Ban et at. 2000), as was a 3 Angstrom structure of the 30S ribosome subunit from T. thermophilus (Wimberly et al. 2000). Again, aspects of structure not visible, even in these atomic resolution structures of the subunits (such as L11, see Wimberly et al. 2000) are visible for the first time in the 5.5 Angstrom 70S structure we describe below. In addition, certain features of the 30S subunit, such as the orientation of the head and platform, differ between the isolated subunits and 70S ribosome.
Crystallization of Thermus thermophilus 70S ribosomes and ribosome complexes (Trakhanov et al. 1987; Trakhanov et al. 1989; Hansen et al. 1990; Yusupova et al. 1991; Yusupov et al. 1991) has provided the possibility for solving the structure of the complete ribosome in different functional states. In an earlier work, we reported the crystallization of functional complexes of the complete T. thermophilus 70S ribosome, containing mRNA and tRNA or tRNA analogs, and the solution of their structures by x-ray crystallography at up to 7.8 Å resolution (Cate et al. 1999). Many specific features of the rRNA were identified, and in many instances, elements of protein structure were also recognizable. The interactions of tRNA with the ribosome in the A, P, and E sites were seen in the greatest detail so far obtained, providing new insights into the mechanism of translation.
Despite these improvements in structure determination of 70S ribosome structure, certain details of the molecular interactions such as those in the interface between the 30S and 50S subunits were not clearly resolved in prior art structure determinations of the 70S ribosome. Knowledge of the details of this and other structural features of the 70S ribosome provides deeper insight into the ribosome function, as well as a structural basis for rational design of novel compounds to alter ribosome function. Thus there exists a need in the art for a higher resolution structure of the 70S ribosome. The present invention provides for these and other advantages by extending the resolution of the 70S ribosome structure to 5.5 Angstroms. Using methods described below, the 5.5 Angstrom structure provides a basis for obtaining high-resolution structural details of the 70S ribosome structure, including determination of many features not previously resolved in prior art structure determinations of the 70S ribosome or its subunits.